Multi-omic profiling converges on proteasome subunits PSMA7/PSMB2 as targets of the sepsis-protective agent Handelin.

INTRODUCTION: Sepsis is a life-threatening systemic inflammatory syndrome with limited targeted therapeutic options. Handelin, a natural compound derived from Chrysanthemum indicum, exhibits anti-inflammatory activity, yet its direct targets and protective mechanisms in sepsis remain unclear. METHODS: We established a lipopolysaccharide (LPS)-induced sepsis-like model in zebrafish larvae to evaluate the protective effects of Handelin. Survival, locomotor behavior, macrophage recruitment, and systemic reactive oxygen species (ROS) levels were assessed. To identify direct protein targets, we performed drug affinity responsive target stability profiling using data-independent acquisition mass spectrometry (DIA-CETSA) in macrophages. Clinical relevance was examined via 4D-DIA proteomics of plasma from a sepsis patient cohort and meta-analysis of public transcriptomic datasets. Molecular docking and dynamics simulations were used to characterize binding interactions. RESULTS: Handelin significantly improved survival, restored locomotor activity, suppressed macrophage aggregation, and reduced ROS in zebrafish. DIA-CETSA revealed that Handelin specifically stabilized multiple core subunits of the 26S proteasome, most notably PSMA7 and PSMB2. In sepsis patients, higher plasma levels of PSMA7 and PSMB2 were associated with increased 90-day mortality and positively correlated with markers of liver injury and SOFA scores. Transcriptomic meta-analysis across 10 independent cohorts revealed that PSMA7 expression was significantly higher in non-survivors than survivors, while PSMB2 showed a similar trend that was marginally significant and sensitive to cohort composition. These findings highlight the complex relationship between transcriptional regulation and clinical outcomes in sepsis, with favorable predicted binding affinities. DISCUSSION: This study suggests that the protective effect of Handelin in sepsis may be associated with its stabilization of the core proteasome subunits PSMA7 and PSMB2. These findings provide new pharmacological insights into the potential anti-septic application of Handelin and propose a novel strategic direction for the treatment of sepsis through precise modulation of proteasome function.